The present invention relates to a cloned and sequenced ECO RI fragment of Bordetella pertussis chromosomal DNA containing the genes which code for the five subunits of the pertussis toxin, useful for the preparation of the pertussis toxin or of one or more subunits of the pertussis toxin.
The present invention also relates to a hybrid plasmid containing the cloned and sequenced DNA fragment or further fragments thereof and to a micro-organism transformed by the hybrid plasmid and capable of expressing the cloned DNA fragment or further fragments thereof by synthesis of the pertussis toxin or one or more subunits of the pertussis toxin.
The invention also concerns a method for the preparation of the pertussis toxin or one or more subunits of the pertussis toxin which includes the growth of the micro-organism transformed by the hybrid plasmid in a suitable culture medium.
The pertussis toxin or one or more subunits of the pertussis toxin thus obtained is useful for the preparation of vaccines and diagnostic kits.
Pertussis is an infection of the respiratory tract caused by Bordetella pertussis (B. pertussis), a Gram-negative coccobacillus which is transmitted directly through the air during a catarrhal or conclusive period from the infirmed to a susceptible healthy individual.
Pertussis may cause respiratory complications, nerve damage and high mortality, particularly in children in low socio-economic groups and in new born babies without maternal, anti-pertussis antibodies. The clinical course of pertussis includes four phases: incubation, cattarhal phase, paroxysmic phase, and a convalescent phase.
During the first two phases there are symptoms comparable to those of a common cold and the B. pertussis may be isolated easily from the patients.
During the paroxysmic phase, characterised by the symptoms of pertussis itself, the bacterium is isolated only in 50% of cases.
During the convalescent phase it is no longer possible to isolate B. pertussis from the nasopharynx although the patients still have symptoms of pertussis.
It is clear from this that the more violent clinical indications of the illness occur after the disappearance of the bacteria and from this it may be inferred that pertussis is not due to invasion of the respiratory tract by the bacterial but to a toxic state induced by the bacteria but which remains even after their disappearance.
The charge of B. pertussis from phase I (virulent) to phase III (non-virulent is accomplished by a loss of capacity to synthesize certain substances such as: the pertussis toxin (PT), haemolysin (hly), adenylcyclase (Adc) and the dermonecrotic toxin (Dmt).
Tests carried out be Munoz. J. J. et al. (1981) (Inf. Immun. 32. 243) have shown that a vaccine constituted by the pertussis toxin alone, suitably detoxified with glutaraldehyde, is capable of protecting mice from death due to the intracerebral administration of bacteria in phase I.
Recent studies (Weiss, A. A. et al. (1983) Inf. Immun 42, 33; Weiss, A. A. et al (1984) J. Inf. Dis. 150, 219) have shown that not all these five substances contribute with equal effect to the virulence of B. pertussis. Weiss has succeeded in isolating the mutants which have lost selectively only one of the factors of the virulence by the insertion of a transposable element, a transposon (Tn5), into the genome of B. pertussis. From tests carried out in animals, it was found that only the mutants which had lost their capacity to synthesize PT or Adc had, at the same time, lost their virulence.
Hence the pertussis toxin (PT) is the major factor in the virulence of Bordetella pertussis. 
The pertussis toxin a protein with a molecular weight of about 100,000 dalton, is produced and released into the extra cellular environment by Bordetella pertussis during phase I.
PT has an enzymatic activity and deactivates ADP-ribosilandol, a GTP-dependent protein which is involved in the deactivation of cellular adenylcylase.
Like other toxins, the pertussis toxin is also constituted by two different fragments: A and B.
The A fragment, which is toxic, comprises a single polypeptide S1 (subunit S1) having a molecular weight of about 28,000 daltons, which can bind an ADP-ribose group to a GTP-binding protein G, which inhibits adenylate cyclase involved in the transmission of signals from the outside to the inside of cells.
The B fragment comprises five polypeptides S2, S3, S4 and S5 (subunits S2, S3, S4, S5) with molecular weights of 23,000, 22,000, 12,000 and 9,000 daltons respectively, disposed as two dimers S2+S4 and S3+S4 and a monomer S5.
The B fragment binds to measure receptors of eucaryotic cells facilitating entry of S1 into the cells.
At present a pertussis vaccine is used which, although giving permanent immunity, has numerous disadvantages.
The vaccine is in fact constituted by virulent bacteria (phase I) treated at 56xc2x0 C. for 30 minutes to remove a toxin which is heat-labile (dermonecrotic toxin) and killed by merthiolate.
Since the bacteria are not subjected to any detoxification treatment, any toxic substance which withstands 56xc2x0 C. for 30 minutes is included in the vaccine.
The presence of such toxic substances, particularly from the PT, causes side effects which vary from simple flushing to permanent neurological damage and/or death.
All this has meant that over the last ten years the use of the vaccine has been reduced drastically with a consequent re-explosion of cases of pertussis.
Recently a vaccine has been prepared which is constituted essentially by fibrous haemagglutinin (FHA) and pertussis toxin detoxified with formaldehyde (Sato Y., et al: Lancet Jan. 21. 122 (1984)).
However, this vaccine has disadvantages such as: the presence of side effects, even though less than those of the conventional vaccine; obtaining a product which is too crude to be used such; and the extreme variability of the product from preparation to preparation.
There is thus a need to provide an effective vaccine which can be produced on a large scale and which does not have the disadvantages noted above.
Thus, for example, recent developments in the biochemical filed and in the field of genetic engineering have made it possible to prepare synthetic vaccines and micro-organisms capable of producing proteins useful for the preparation of vaccines with high yields.
In every case a key element for the preparation of the vaccines is a knowledge of the amino acid sequence of the protein and the nucleotide sequence of the gene and/or genes which code for the protein.
Once the gene which codes for a certain protein has been cloned and its nucleotide and amino acid sequences have been determined, the production of these on a large scale and the construction of synthetic vaccines is possible with current techniques.
At present nothing is known of the nature, structure and expression of the gene and/or genes of the pertussis toxin and no data other than the amino acid composition of the individual subunits of the pertussis toxin is available.
Accordingly, by the present invention there has been determined the aminoterminal amino acid sequence of the subunits S1, S2, S3 and S4 of the pertussis toxin and an Eco-RI-fragment of Bordetella pertussis chromosomal DNA has been cloned and sequenced, the fragment having 4696 base pairs and containing the genes which code for the five subunits of the pertussis toxin, useful for the preparation of the pertussis toxin or of one or more subunits of the pertussis toxin. Thus a subject of the present invention is a cloned and sequenced 4696-base-pair Eco RI fragment of Bordetella pertussis chromosomal DNA containing the genes which code for the five subunits of the pertussis toxin or fragments thereof, useful for the production of the pertussis toxin or of one or more subunits of the pertussis toxin.
Another subject of the invention is a hybrid plasmid containing the cloned and sequenced DNA fragment or further fragments thereof.
A further subject of the present invention is a micro-organism transformed by the hybrid plasmid and capable of expressing the cloned DNA fragment or its further fragments by synthesis of the pertussis toxin or of one or more subunits of the pertussis toxin.
Another subject of the present invention is a method for
Another subject of the present invention is a method for the preparation of the pertussis toxin or of one or more subunits of the pertussis toxin by growth of the transformed micro-organism.
A further subject of the present invention is the use of the pertussis toxin or of one or more subunits of the pertussis toxin for the preparation of anti-pertussis vaccines and diagnostic kits.
Yet another subject of the invention is the protein of the pertussis toxin in which the subunits S1, S2, S3, S4 have the amino acid sequences given in FIGS. 2 and 3. Further subjects of the present invention will become apparent from the description and the experimental examples which follow.
Genetic Code: by this term is meant the relationship existing between the nucleotide sequence in DNA and the amino acid sequence in a protein.
An important characteristic of the genetic code is the fact that the synthesis of each amino acid is specified by a sequence of three nucleotides in the DNA, also called a triplet or condon.
The genetic code is universal, that is, a particular triplet codes the same amino acid in all living beings.
Reading phase or frame: by this term is meant a group of triplets used by a cell to decode the genetic message.
Cloning vectors: these are molecules of DNA which contain all the genetic information to enable them to replicate when transferred into a host micro-organism.
Examples of cloning vectors commonly used in genetic engineering are the plasmids and the DNA of several bacteriophages.
The plasmid DNA, which is circular, may be cut by suitable techniques and a heterologous DNA fragment may be inserted and the ring reclosed to form a larger molecule containing the heterologous DNA, the so-called molecule of recombinant DNA or hybrid plasmid.
The DNA of bacteriophage may contain a segment of heterologous DNA inserted instead of several non-essential genes. Both these vectors are used for the insertion of heterologous DNA fragments and for the subsequent transformation of micro-organisms, also called host cells.
Restriction enzymes: these are hydrolytic enzymes capable of cutting a DNA molecule at specific sites, so-called recognition sites for the restriction enzymes.
Transposons: these are segments of DNA which may transpose and insert themselves at different points in the genome and give rise to the process known as transposition.
Promoter: a specific region of the DNA molecule in which the RNA polymerase starts transcription.
The promoter includes a recognition site and a binding site for the enzyme.
Termination Region: a specific region of the DNA molecule in which transcription ends.
Translation: this is the passage of genetic information from the mRNA to the protein according to the rules of the genetic code.
Expression: this term means the mechanism by means of which an organism can synthesise a protein coded by a specific gene.
In this case one says that the gene is expressed by the micro-organism.
In general, a method for obtaining a heterologous protein by recombinant DNA techniques requires the cloning of the gene which codes for the protein, where by cloning is meant the sequencing, isolation and purification of the gene and/or genes which code for the protein. Once cloned, the gene may be inserted in an expression vector and the molecule of recombinant DNA thus obtained may then be introduced into a host micro-organism where the gene will replicate simultaneously with the replication of the micro-organism, from which it may be re-isolated by conventional methods.
With this method of operation it is possible to provide a continuously renewable source of the gene which can then be manipulated further, modified and inserted in other vectors or in different sites in the same vector.
The transformed micro-organism, grown in a suitable culture medium, will enable the protein coded by the gene to be synthesized.
Accordingly by the present invention there has been cloned and sequenced an Eco RI fragment of Bordetella pertussis BP 165 chromosomal DNA containing the genes which code for the five subunits of the pertussis toxin and the aminoterminal sequence of the subunits S1, S2, S3 and S4 of the pertussis toxin has been determined. In particular, the pertussis toxin produced by Bordetella pertussis 165 has been purified by affinity chromatography and the subunits subsequently separated by electrophoresis in polyacrylamide sodium dodecylsulphate gels as shown in FIG. 1.
The individual subunits were then separated and purified by electroelution (Hunkapiller M. W. et al.; Methods in Enzymology 91, 227-236, 1983) and analysed in a gas-phase microsequencer.
The aminoterminal sequence of the subunits S1, S2, S3 and S4 is given in FIG. 2.
A gene library was then constructed with the use of the E. Coli lambda phage EMBL4 (bought from Promega Biotec 280 S. Fish Hatchery Road, Madison, Wis. 53711 USA) starting from the strain Bordetella pertussis BP356.
This strain is a mutant which does not produce an active toxin and has a single transposon TN5 inserted into its chromosome [Weiss, A. A. et al. Infect. Immun. 42, 33-41 (1983)].
The chromosomal DNA of the said strain was separated from the cells and, after purification, was partially digested with the restriction enzyme Sau3A1 by the method and under the operative conditions described by Maniatis T. et al.: Molecular Cloning a Laboratory Manual Cold Spring Harbor N.Y., (1982). The fragments of chromosomal DNA with 15000 to 20000 base pairs were then separated and cloned in the E. coli lambda phage vector EMBL4 previously prepared as reported by Frischauf A. et al. [J. Mol. Biol. 170, 827-842 (1983)] with the use of the Promega Biotec xe2x80x9cPackagenexe2x80x9d Kit according to the method described by Maniatis T. et al. (Molecular Cloning a Laboratory Manual Cold Spring Harbor N.Y. 1982).
The recombinant phages were then used to transform E. coli NNM 539 cells Promega Biotec).
The phages containing DNA fragments in which the transposon TN5 had been inserted were then selected from the transformed cells by the plate-hybridization technique with a radio-active probe for the TN5 DNA.
The recombinant phage DNA was then extracted from the positive recombinant phages and, after digestion with the restriction enzyme Eco-RI, the DNA fragments containing the transposon TN5 were separated and selected by hybridization with a probe for TN5 DNA.
In this manner it was possible to isolate an Eco-RI DNA fragment with about 10500 base pairs containing the entire sequence of the transposon TN5 flanked on the one hand by about 1100 base pairs and on the other by about 3500 base pairs of chromosomal DNA of Bordetella pertussis BP 356.
The Eco-RI fragments with 10500 base pairs were then digested with the restriction enzyme Hinc II and the DNA fragments containing the junction between the TN5 and the chromosomal DNA were isolated by hybridization with a probe for TN5 DNA.
Two fragments were thus identified, one with about 500 base pairs and the other with 1900 base pairs.
The two fragments, purified by electroelution, were then cloned in the phage vector M13mp8 (New England Biolabs) the DNA thereof had previously been cut but the restriction enzyme Hinc II.
The nucleotide sequences of the two fragments were then determined, starting from the Hinc II site according to the technique described by Sanger F. S.: Proc. Natl. Acad. Sci. 74, 5463 (1977).
The fragment with 1900 base pairs had at about 400 nucleotides from the Hinc II site, a nucleotide sequence (FIG. 3A from 3030 to 3100 bp) which, translated into the corresponding amino acids according to the genetic code, corresponded exactly to the amino acid sequence determined previously for the subunit S3 and given in FIG. 2.
This result confirms that the cloned DNA fragment with 10500 base pairs contained the gene for the pertussis toxin.
The fragment with 1900 bp was then used as a hybridization probe to identify and isolate a fragment DNA fragment containing the gene for and/or which codes for the pertussis toxin from the chromosomal DNA of B. pertussis EP 165 for which a gene library had been constructed as described above for B. pertussis BP 356.
At the end of the cloning operations, a 4696 base-pair Eco RI fragment of chromosomal DNA was isolated which we knew contained at least the gene which codes for the subunit S3 in that the fragment hybridized with the specific probe for S3.
The said fragment or parts thereof were then cloned in the phage vector M13mp8 and M13mp9 and the recombinant phage DNA thus obtained was sequenced.
Analysis of the sequence has enabled various open reading frames (ORFS) to be identified.
A comparison of their coding properties and the amino-terminal sequences of the subunits of the toxin have shown that four of these ORFS in fact code for the subunits S1, S2, S3 and S4 of the pertussis toxin.
Moreover, the molecular weight, the amino acid composition and the electric charges were in exact accordance with published data (Table 1) A fifth ORFS was also identified, placed between those which code for S4 and S3, which codes for a protein with a molecular weight and an amino acid composition identical to those described for the subunit S5.
These five open reading frames are grouped in a fragment, with 3200 base pairs in the following order: S1, S2, S4, S5 and S3 and the ORFS reading frame which codes for S4 is superposed on those which code for S2 and S3 (FIG. 3). On the basis of these results it is possible to conclude that the sequences determined contain the genes which code for the subunits of the pertussis toxin, and hence the open reading frames will be termed genes below.
In accordance with the present invention a transcription signal, very similar to the concensus sequence for the E. coli promotors, was identified before the gene which codes for S1.
In fact a regionxe2x80x9410, TAAAAT, which contains five of the six base pairs of the concensus sequence is associated with a regionxe2x80x9435, TGCTGACC, which contains six of the eight bases of the concensus sequencexe2x80x9435.
The distance between the two regionsxe2x80x9435 and xe2x80x9410 is 21 base pairs.
At the end 3xe2x80x2 of the gene which codes for S3 there has been identified an inverted repeated sequence followed by a poly-T sequence which could represent a termination site.
Since no other promoter before the four genes S2, S3, S4 and S5 has been identified in the DNA fragment it may be deduced that these genes are organized in a single operon and are transcribed as a single polycistronic mRNA.
The presence of a single Shine-Dalgarno sequence located nine base-pairs before the ATG of the gene S1, strongly suggests that this is the ribosomal binding site which enables the translation of the S1 mRNA.
The presence of a new consensus sequence, TCC (T) GG, located eight to twelve base pairs before each ATG initiation codon for the four genes, suggests that this site is responsible for the translation of the entire mRNA.
Moreover it was found that the gene S4, which is produced in stoichiometric quantities of 2 to 1 with respect to the other genes, is the only one which is preceded by a slightly modified consensus sequence, TCCTG, which probably increases the translation efficiency.
A characteristic common to all the subunits of the pertussis toxin is the presence, in the gene, of a sequence immediately preceding the mature protein, which codes for a 27-42 amino acid peptide the characteristics of which are typical of signal peptides involved in the secretion of the proteins.
This suggests that the various subunits are synthesized as proproteins, processed and secreted individually in the periplasmic space and subsequently processed, assembled and released into the extra-cellular space in the form of a single protein.
It has also been found that the signal peptide for S4 is unexpectedly long (42 amino acids) and has the highest aminoterminal positive charged described until now.
Since the positively-charged aminoterminal regions play an important role in the efficiency of production of the secreted proteins, the unusual structure of the signal peptide for S4 could cause increased translation of the gene S4.
It was also noted that, in the absence of the subunit S3 as occurs in the mutant BP356, the pertussis toxin is not excreted into the culture medium. Consequently, this protein is necessary for the complete assembly of the pertussis toxin.
The cloned DNA fragment or further fragments thereof, the said fragments containing at least one gene which codes for at least one subunit of the pertussis toxin, must be capable of being inserted in an expression vector and the hybrid plasmid thus obtained may be used to transform a micro-organism.
The transformed micro-organisms, grown in a suitable culture medium, are able to express the DNA fragment or fragments thereof by synthesis of the pertussis toxin or one or more subunits of the pertussis toxin.
Cloning vectors suitable for the purpose may be selected from natural plasmids known in the art or synthetic vectors obtained by recombinant DNA techniques.
In particular, the plasmid of E. coli pEMBL8 with about 4000 base pairs is used, this containing the gene for resistance to ampicillin and restriction sites useful for the cloning, such as: HindIII, pstI, AccI, HincII, SalI, BamHi, AvaI, SmaI, Xmai, EcoRI (Dente L. et al (Nucleic Acids Research 11, 1645-1655 (1983)), and the plasmids 31A, 31B and 31C derived from the vector PEX29 (Klinert M. et al. Inf. Imm. 49, 329-335 (1985)) which contain the gene which codes for the DNA polymerase of the phage MS2 placed under the control of the inducible promoter pL and a polylinker inserted before the end of the gene of the MS2 polymerase in three possible frames, so as to be able to break each possible DNA fragment in the same frame of the MS2 poymerase.
Examples of micro-organisms used as host cells are strains of Escherichia coli, Bacillus subtilis, Saccharomyces, or eucaryotic cells.
In accordance with the present invention, there are used cells of E. coli JM 101 (New England Biolabs 32 Tozer Road, Beverly, Mass. 01915-9990 USA) and cells of E. coli K-12 H1trp (described by Remant E. Gene 15: 81-93 (1981)) which produce a heat-sensitive repressor which, at 30xc2x0, completely inhibits the transcription of the gene of the MS2 polymerase preventing the production of proteins fused to it and, at 42xc2x0 C., is inactivated giving good production of the polymerase and of the proteins fused to it.
The choice of the cloning vector and of the micro-organism to be transformed are not however limited by the present invention.
In accordance with the present invention, the 4696 base-pair fragment of chromosomal DNA obtained as described above, was inserted in the plasmid vector of E. coli pEMBL-8 after digestion of the plasmid DNA with the restriction enzyme Eco RI.
The hybrid plasmid obtained, designated pPT101, was then used to transform cells of E. coli JM101 (New England Biolabs) made competent by the method described by Cohen S. et al. (Proc. Natl. Acad. Sci. U.S. 69, 2110 (1972)).
The strain of E. coli (pPt101) was deposited in the American Type Culture Collection on Jun. 8, 1985 with the number ATCC 67854 as a substitute for ATCC 53212.
In order to check the ability of the transformed micro-organism to express the cloned DNA, fragment, the E. coli strain (pPT101) was cultivated in a suitable culture medium.
More particularly, the strain was grown in LB medium (DIFCO) at a temperature of 37xc2x0 C. up to an absorbance of 0.75, measured in the culture broth at 590 nm.
The cells were then subjected to lysis and the pertussis toxin was determined directly in the cellular lysate by immunoenzymatic methods.
The biological activity of the pertussis toxin was determined by the method reported by Hewlett E. L. et al. (1983) (Infect. Immun. 40, 1198-1203), the change in form of the CHO cells incubated with the cellular lysate under examination being analysed.
The results obtained confirm that the 4696 base-pair fragment of Bordetella pertussis chromosomal DNA contains the genes which code for the five subunits of the pertussis toxin and the said toxin can be neutralized by antibodies against the toxin itself.
According to one embodiment of the present invention, the genes which code for the individual subunits of PT were cloned in the plasmids 31A, 31B, 31C derived from the vector PEX29 and the hybrid plasmids thus obtained and designated PTE255 (S1), PTE211 (S2), PTE221 (S3), PTE240 (S4) and PTE230 (S5) were used to transform cells of E. coli K-12 H1 trp.
The cells thus transformed were then cultivated in a suitable culture medium and the subunits, obtained as fused proteins, were recovered, purified and tested to determine their biological activities.
The results obtained show that all five subunits, when injected into rabbits, induce the formation of specific antibodies.
Moreover, the fused S1 protein shows the same enzymatic activity as the entire PT toxin, thus showing not only an immunological but also a functional identity with the natural S1.
In fact ADP-ribosylation tests carried out by incubating fused S1 with homogenized ox retina (ROS) in the presence of NAD marked with 32 P, indicate that the subunit S1 binds the ADP-ribose group to the transducine present in the retina.
Hence both the pertussis toxin and the individual subunits obtained by the method of the present invention may be used for the preparation of vaccines against pertussis and diagnostic kits for determining specific antibodies in clinical samples from individuals infected with pertussis.
Analysis of the sequences given in the present invention also shows a certain similarity between the amino acid sequence in the subunit S1 of the pertussis toxin and that of the subunit A of the cholera toxin (J. Mekalanos et al. Nature 306, 551-557, 1983) (FIG. 7).
There is thus a possibility of preparing a vaccine capable of neutralizing cholera and pertussis simultaneously, with the use of the peptide S1 made by chemical synthesis or by recombinant DNA techniques.